Cellular enzymes are a key to metabolic diversity, differentiation and cytopathology among cells. However, cellular enzymes are extremely diverse. Their functions include host defense, transport of molecules through membranes, production of energy and synthesis of the cellular constituents. As many as a thousand different enzymes might be operative in any given cell, but only a few dozen may define the function of any one cell type. In addition, enzyme levels can vary by factors of tens to hundreds, depending on the functional or differential state of the cell. Furthermore, these same enzymes may be non-detectable in quiescent cells of the same functional phenotype and cells performing different functions.
Morphologic classification of chemical constituents within cells was aided by the use of cytochemical stains. These methods used were enzymatic techniques and in general, all prior art assays for enzymatic activity have been cytochemical colorimetric assays. Examples of the prior art measurement of enzyme activity are:
1) Acetate esterase activity measured with .alpha.-Napthyl acetate has been used together with other esterase activities to identify leukocyte cell types and is generally high in normal monocytes and megakaryocytes and in blast cells of acute myelomonocytic leukemia, acute monocytic leukemia and acute erythroleukemia.
2) Cloroacetate esterase activity measured with Naphthol-AS-D chloroacetate is generally high in normal promyelocytes and neutrophils and in blast cells of acute myeloblastic leukemia with maturation, acute promyelocytic leukemia and acute myelomonocytic leukemia.
3) Butyryl esterase activity measured with .alpha.-Napthyl butyrate has been used to identify different cell types and is generally high in normal monocytes and in blast cells of acute myelomonocytic leukemia and acute monocytic leukemia. Butyryl fluorescein is also a substrate for phospholipase A.sub.2, an early enzyme in the biochemical cascade leading to the production of prostaglandins and leukotrienes.
4) Assays of acid phosphatase activity have been used together with assays of esterase activity to identify many different cell types. Monocytes, neutrophils and T-lymphocytes have relatively high acid phosphatase activity while B-lymphocytes have relatively low acid phosphatase activity. In addition, blast cells of acute promyelocytic leukemia and acute myelomonocytic leukemia have been shown to have relatively high acid phosphatase activity.
5) A derivative of .beta.-glucuronidase has been used to measure degranulation in polymorphonuclear lymphocytes (PMN) in a test of the ability of different non-steroidal anti-inflammatory drugs (NSAIDS) to inhibit PMN functions. Peripheral blood T-lymphocytes display higher .beta.-glucuronidase activity that peripheral blood B-lymphocytes. Fluorescein di-glucuronide is a negatively charged compound. To help other derivatives of sugars pass through cell membranes in assays of .beta.-glucosidase, a lysomotropic detergent (N-dodecylimidazole) was used.
The study of enzymes by flow cytometry may have had its beginnings in 1957 when Lowry (J. Bio. Chem., 224:1047-1067 (1957)) studied dehydrogenases by fluorescence microscopy in cells using NADP. Rotman, (J. Immunology, Vol. 120, No. 8, pp. 1460-1464 (1978); PNAS USA, Vol. 75, No. 2, pp. 720-724 (1978); and PNAS, Vol. 60, pp. 660-667 (1968)) studied .beta.-galactosidase in ribosomes. In 1969, Hulett (Science, 166:747-749 (1969)) prepared esterase compounds with fluorescein. Naphthylamine, naphthol and coumarin derivatives were studied by Dolbeare and Smith (Clin. Chem. 23/8,1485-1491 (1977)). Functional cell assays for ionized calcium, intracellular pH, glutathione and membrane potential were studied by Rabinovitch (NYAS, 667:252 (1990) and Valet (NYAS, 677:233 (1993)) described phagocytosis, respiratory burst, activation antigens and protease activity in leukocytes.
Dichlorofluorescin diacetate (2', 7' dicholorofluorescin diacetate hereinafter referred to as DCFH-DA) as a cellular substrate for oxidative burst was first suggested by Bass, et. al. J. Immunology, Vol. 130, No. 4, pp. 1910-1917 (1983). It is sensitive to the oxygen radical. The use of DCFH-DA was used to determine oxidative burst resulting from peroxidase or catalase in neutrophil cells. The concept was that a reaction with DCGFH-DA provided a functional test of a neutrophil cell to determine whether or not there was enough oxygen radical to neutralize bacteria. Therefore, the test of neutrophil functionality was to determine its efficacy in fighting disease.
Bass et al. J. Immunology, Vol. 130, No. 4, pp. 1910-1917 (1983), first monitored the oxidative burst in neutrophils using DCFH-DA in 1983. Bass et al. proposed that the conversion of non-fluorescent dichlorofluorescin diacetate (DCFH-DA) to the highly fluorescent compound 2',7'-dichlorofluorescein (DCF) happens in several steps. First, DCFH-DA is transported across the ceil membrane and deacetylated by esterases to form the non-fluorescent compound 2',7'-dichloroflluorescin (DCFH). This compound is trapped inside cells. Next, DCFH is converted to DCF through the action of peroxide (H.sub.2 O.sub.2). ##STR1##
Measurement of the fluorescence of the DCF is therefore a measure of the production of H.sub.2 O.sub.2.
The contribution of peroxidase to the oxidation of DCFH is unknown. In solution, the oxidation of DCFH is markedly increased by peroxidase. However, azide, which is an inhibitor of myeloperoxidase, also increases intracellular oxidation of DCFH. Bass et al. hypothesize that this increase may be due to azide-mediated inhibition of catalase, an enzyme that competes with DCFH for interaction with peroxide generated by the cell.
Rothe, Oser and Valet, Naturwissenschaften, 75, 354-355 (1988), introduced dihydrorhodamine 123 (DHR 123) as a flow cytometric indicator for oxidative burst activity in neutrophils in 1988 as a more sensitive probe of oxidative burst activity than DCFH-DA. Rothe, Oser and Valet found that DHR 123 could detect the relatively small oxidative burst increase produced by stimuli such as the chemotactic peptide f-Met-Leu-Phe; this increase was only barely measurable with DCFH-DA.
Both DCFH-DA and DHR 123 are used to measure products of the oxidative burst of polymorphonuclear leukocytes. During the oxidative burst, cellular enzymes NADPH oxidase and superoxide dismutase produce the superoxide anion (O.sub.2.sup.-) and hydrogen peroxide (H.sub.2 O.sub.2) in the following reactions: ##STR2## When myeloperoxidase is present, it breaks down the hydrogen peroxide: ##STR3##
Royall and Ischiropoulos Arch. Biochem. & Biophys., Vol. 302, No. 2, pp. 348-355 (1993), did several experiments on the permeability of the cell membranes of cultured endothelial cells to DCFH-DA, DCFH, and DCF. In their experiments, Royall and Ischiropoulos incubated cells in media containing DCFH-DA and then washed the cells into media without DCFH-DA. They then measured the intracellular and extracellular concentrations of DCFH-DA, DCFH, and DCF. They found that there was a greater than 90% loss of DCFH and DCF from the cells after one hour, demonstrating that DCFH and DCF are not trapped within the endothelial cells.
In their experiments on the diffusion of the probes across the cell membranes, Royall and Ischiropoulos found that intracellular DHR 123 concentrations, like DCFH and DCF concentrations, decreased by 90% after the cells were incubated for one hour in media that did not contain DHR 123. However, the product of the reaction, intracellular rhodamine 123, decreased by only 15% after 1 hour. Therefore, the rhodamine 123 was found to be retained by the cell better than DCF.
In their experiments on the sensitivity of the probes, Vowells, J. Imm. Methods, 178, pp. 89-97 (1995), et al. found that the fluorescent signal measured in normal granulocytes stimulated with PMA was seven times higher for DHR 123 than DCFH-DA. The addition of 0.017% azide increased the signal from DCFH-DA by 140% and the signal from DHR 123 by 25%. Vowells et al. also studied mixtures of normal granulocytes and granulocytes from patients with chronic granulomatous disease (CGD), a rare genetic disorder caused by defects in the NADRH oxidase enzyme complex. Vowells et al. found that DHR 123, but not DCFH-DA, could detect normal sub-populations as small as 0.1% in mixtures of normal/CGD granulocytes.
The necessity to detect blast cells in a patient sample to determine a treatment regime for leukemia has long been desired. Consequently, to determine blast accurately, one uses a Wright stain, which is a classical staining technique to confirm the presence of blast cells and identifies cells by their lipid protein and nucleic acid concentrations. Then, one employs a colorimetric peroxidase stain on a microscope slide preparation to indicate the presence or absence of the peroxidase in the blast cell. A panel of cytochemical stains are employed to microscopically identify these malignant cell types. The use of these specific staining techniques can then establish the cell line involved. In these techniques, the cells are not metabolically active.
Over the years, the microscopic examination of leukocytes, erythrocytes and platelets on a blood film, known as the manual differential count, has been recognized as the foundation for diagnosis of hematological abnormalities. However, the manual method has the reputation of being expensive and tedious, requiring a highly skilled technologist and having a relatively high inherent error rate. The manual method's high variability is related to the preparation techniques, sample size, and operator subjectivity.
In addition, acute leukemia patients are often treated with drugs which have effects upon bone marrow cell production roles and which can alter the morphology of blood cells, making it difficult to correctly identify these cells. Results are based on subjective interpretations.
Clinical practice would prefer determining blast cells at less than 1%. Early detection is most preferable because treatments, consisting of chemotherapy and other harsh treatments which kills normal cells as well as abnormal cells, could be administered to minimize the adverse consequences of the treatment and a lower detection level will also enable the monitoring the efficacy of drug therapies.